This project is designed to provide new information on the biochemical basis for virulence in Mycoplasma pneumoniae infections, particularly Primary Atypical Pneumonia. We will study the molecular mechanisms of pathogenesis through an analysis of receptor site interactions and host cell nucleic acid metabolism. Receptor site studies will begin with membrane fractions prepared from human lung fibroblasts by using the zinc/fluorescein mercuric acid technique. Glycoproteins will be extracted by 3 methods: lithium diiodosalycilate, TRIS/EDTA, and Triton x-100. Glycoproteins will be quantitated with gradient (7.5 to 20 %) polyacrylamide gel electrophoresis and silver nitrate/dansyl hydrazine staining. The major species will be isolated by column chromatography (Bio Gel P-150) and preparative polyacrylamide gel electrophoresis. Each will be evaluated for its ability to inhibit attachment of 14C-labelled mycoplasmas to lung fibroblast monolayers and to hamster tracheal explants. The receptor will be characterized relative to molecular weight, carbohydrate and amino acid composition. The effect on tunicamycin on receptor biosynthesis will be evaluated. Because a major effect on host cells post-attachment is a decrease in purine synthesis, we will also study the nucleic acid biochemistry of infected cells. High-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) will be used to quantitate purine nucleotides in control and infected cells (fibroblasts and tracheal explants). The influence of mycoplasma infection on lung fibroblast adenosine deaminase activity will be evaluated with a TLC assay. We will also evaluate the ability of deoxycorformycin to potentiate cytotoxicity by inhibiting adenosine deaminase. These data will indicate where Mycoplasma pneumoniae disrupts the metabolic pathway of de novo purine synthesis. This information on attachment and mechanism of action will be used to delineate the infective process, and provide a new perspective on which to base improved therapy and prophylaxis.